Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in the huntingtin (Htt) gene. Mutant Htt forms intracellular aggregates and is cytotoxic to specific neurons in the striatum and cortex. Causes of cell death in HD include the activation of cell death genes and/or alteration of normal transcription. The objective of this proposal is to investigate how mutant Htt interacts with NF-kappaB signaling pathway. This pathway regulates expression of pro-survival genes, is central to the function of neurotrophic factors and cytokines, and is activated during excitotoxic cell death mediated by NMDA receptors. We are investigating the interaction of mutant Htt with the IkappaB kinase (IKK) complex that regulates NF-kappaB, and how this affects neuronal responses to extracellular signals. We will focus on the mechanism of how the direct interaction between mutant Htt and IKKgamma, a regulatory component of IKK, influences neurodegeneration in HD. Molecular, cell culture, brain slice and animal model approaches will help to explore the nature and consequence of this interaction. We have preliminary evidence that IKKgamma influences NF- kappaB-regulated neuronal gene expression, as well as mutant Htt-induced cell death and aggregation. (i) Using in vitro and cellular assays, we are mapping the domains in mutant Htt and IKKgamma that mediate their binding. (ii) The functional consequences of Htt-IKKgamma interaction are assessed by isolating IKK complex from mutant and normal Htt-expressing cultured cells and HD transgenic mouse brain. Its kinase activity and ability to regulated NF- kappaB mediated gene expression is then tested using in vitro kinase and gene reporter assays. (iii) Downstream genes influenced by the Htt-IKKgamma interaction will be identified by cDNA mini-arrays. mRNA will be isolated from several brain regions from HD and normal mice. (iv) The influence of IKKgamma on mutant Htt-induced cell death and aggregation is being examined in HD transgenic mice and in PC12 cells that inducibly express Htt and/or IKKgamma. We have constructed lentiviral vectors expressing a dominant negative form of IKKgamma as well as cell permeable peptides that block IKKgamma activity in cells. These reagents will be injected into the brains of HD mice in tests of their efficacy in inhibiting mutant Htt toxicity and aggregation. They will also be tested on brain slices from HD mice, in the presence and absence of NMDA.